Dale D. Hunter

Globose basal cells are neuronal progenitors in the olfactory epithelium: A lineage analysis using a replication-incompetent retrovirus

We have used a replication-incompetent retrovirus to analyze the lineage of olfactory receptor neurons in young rats. At 5-40 days after infection, clusters of infected cells comprised two major types: one consisted of 1-2 horizontal basal cells, and a second consisted of variable numbers of globose basal cells and immature and mature sensory neurons. Olfactory nerve lesion (which enhances neuronal turnover) increased the frequency of the globose-sensory neuron clusters as well as the number of cells in such clusters. No clusters contained both horizontal and globose basal cells, and none contained sustentacular cells. These data suggest, at least in young rats, that horizontal basal cells are not precursors of olfactory neurons, that there is a lineage path from globose cells to mature neurons, and that sustentacular cells may arise from a separate lineage.

S-laminin expression in adult and developing retinae: A potential cue for photoreceptor morphogenesis

The development of the neural retina follows a stereotyped time course that begins with an undifferentiated neuroepithelium populated by multipotential progenitor cells and ends with a highly differentiated tissue containing diverse cell types. The identities of the factors that guide this differentiation have remained elusive; a likely location for such factors, however, is the extracellular environment. Here, we show that the extracellular matrix component s-laminin is present in the neural retina, that s-laminin expression parallels the differentiation of rod photoreceptors, that photoreceptors interact with s-laminin in vitro, and that antibodies to s-laminin profoundly reduce the appearance of cells that express rhodopsin in vitro. These data suggest that s-laminin plays a role in the differentiation of the neural retina and provide evidence that the composition of the extracellular matrix may be an important determinant of retinal differentiation.

Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina.

BackgroundThe Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk) family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1) and Dickkopf 2 (Dkk2) are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3) protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death.ResultsDkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis.ConclusionThese data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

The extracellular matrix component WIF-1 is expressed during, and can modulate, retinal development

We have shown previously that components of the extracellular matrix (ECM) modulate neuronal development. Here, we searched for additional ECM elements that might play roles in retinal histogenesis and identified a secreted glycoprotein that is heavily expressed in the retina. This molecule, named by others Wnt Inhibitory Factor-1 (WIF-1), is expressed during and after the period of rod photoreceptor morphogenesis in the mouse. We show that a potential WIF-1 ligand, Wnt4, as well as a potential Wnt4 receptor, fzd4, and a potential Wnt4 coreceptor, LRP6, are expressed in the region of, and at the time of, rod photoreceptor genesis. WIF-1 and Wnt4 are coexpressed during retinal development and bind to each other; therefore, they are likely to interact during rod production. WIF-1 protein inhibits rod production, and anti-WIF-1 antibodies increase rod production; in contrast, Wnt4 promotes rod production. Together, these data suggest that WIF-1 and Wnt4, both components of the ECM, regulate mammalian photoreceptor development.

Collagen XVII and BPAG1 expression in the retina: evidence for an anchoring complex in the central nervous system.

The ectoderm gives rise not only to the skin but also to the entire CNS. This common embryonic lineage suggests that some molecular isoforms might serve analogous functions in both tissues. Indeed, not only are laminins important components of dermal adhesion mechanisms, but they also regulate some aspects of synaptic development in both the CNS and the PNS. In the skin, laminins are part of a hemidesmosome complex essential for basal keratinocyte adhesion that includes collagen XVII (BP180) and BPAG1 (dystonin/BP230). Here, we show that CNS neurons also express collagen XVII and BPAG1 and that these molecules are expressed in the adult and developing retina. In the retina, isoforms of collagen XVII and BPAG1 are colocalized with laminins at photoreceptor synapses and around photoreceptor outer segments; both molecules are expressed by rods, whereas cones express collagen XVII but not BPAG1. Moreover, biochemical data demonstrate that collagen XVII complexes with retinal laminins. We propose that collagen XVII and BPAG1 isoforms may help to anchor elements of the rod photoreceptor cytomatrix to the extracellular matrix. J. Comp. Neurol. 487:190–203, 2005.

Laminins containing the β2 chain modulate the precise organization of CNS synapses

Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate beta2-containing laminins, including perhaps laminins 13 (alpha3beta2gamma3) and 14 (alpha4beta2gamma3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.

5-HT2a receptors in the rabbit retina: potential presynaptic modulators.

Three 5-HT receptors have been implicated in retinal processing but positive identification of the receptors and the localization of receptor subtypes in the retina have not been achieved. In this study, molecular techniques were used to identify one class of 5-HT receptor--5-HT2a--in the retina, and immunohistochemical techniques were used to localize the receptor in the retinal network. Reverse transcription polymerase chain reaction (RT-PCR) techniques were used to identify a segment of the rabbit 5-HT2a gene; a 422 base fragment was identified, cloned, and sequenced. The fragment shows a high degree (ca. 90%) of nucleotide sequence identity with the 5-HT2a receptor gene from other mammals. 5-HT2a immunoreactivity was seen in both the inner and outer plexiform (synaptic) layers of the retina. Using cell-type-specific markers, the 5-HT2a immunoreactivity was shown to be on the terminals of photoreceptor and rod bipolar cells. This association of 5-HT2a receptors with these two synapses suggests that serotonin may be a modulator of synaptic function in the retina.

Laminin deficits induce alterations in the development of dopaminergic neurons in the mouse retina

Genetically modified mice lacking the beta2 laminin chain (beta2null), the gamma3 laminin chain (gamma3 null), or both beta2/gamma3 chains (compound null) were produced. The development of tyrosine hydroxylase (TH) immunoreactive neurons in these mouse lines was studied between birth and postnatal day (P) 20. Compared to wild type mice, no alterations were seen in gamma3 null mice. In beta2 null mice, however, the large, type I TH neurons appeared later in development, were at a lower density and had reduced TH immunoreactivity, although TH process number and size were not altered. In the compound null mouse, the same changes were observed together with reduced TH process outgrowth. Surprisingly, in the smaller, type II TH neurons, TH immunoreactivity was increased in laminin-deficient compared to wild type mice. Other retinal defects we observed were a patchy disruption of the inner limiting retinal basement membrane and a disoriented growth of Müller glial cells. Starburst and AII type amacrine cells were not apparently altered in laminin-deficient relative to wild type mice. We postulate that laminin-dependent developmental signals are conveyed to TH amacrine neurons through intermediate cell types, perhaps the Müller glial cell and/or the retinal ganglion cell.

Roles of the extracellular matrix in retinal development and maintenance.

The extracellular matrix (ECM) is a highly organized meshwork of secreted macromolecules that consists of proteins, proteoglycans, and polysaccharides. Components of the ECM provide cells with different signals that are involved in many aspects of the development of multicellular organisms, such as: (1) when to start, continue, or stop dividing; (2) when to differentiate; (3) where to migrate; (4) how to polarize; (5) where to form a synapse; and (6) whether to die or survive. Thus, ECM molecules may be playing many different roles in the development of a complex tissue such as the retina.

S-laminin and N-acetylgalactosamine located at the synaptic basal lamina of skeletal muscle are involved in synaptic recognition by growing neurites.

SummaryThe purpose of the work reported here is to identify molecular components of the synaptic basal lamina of skeletal muscle fibres which allow recognition of original synaptic sites by regenerating motor axons. We focused on s-larninin and components recognized by the lectinDolichos biflorus agglutinin previously shown to be specifically located at the synaptic basal lamina. We used a cryoculture bioassay in which chick ciliary ganglion neurons grow on rat skeletal muscle cryostat sections. In control cultures, neurites extended over the muscle sections in close association with the muscle cell surface. It was observed that most of the neurites that extended towards the endplate zone and reached an area of 40 μm around the neuromuscular junction ceased to grow when they contacted the synaptic site. Masking either lectin receptors or some s-laminin molecule epitopes prior to the culture of neurons alters the behaviour of growing neurites. On sections treated either withDolichos biflorus agglutinin or anti s-laminin monoclonal antibodies (D5 and C4) most of the neurites did not stop their growth at the synaptic regions. Moreover, treating muscle sections withDolichos biflorus agglutinin removed the gradient of substratum affinity around the endplate. These results indicate that the s-laminin andDolichos biflorus agglutinin receptors present on muscle cell surfaces may play a functional role in the interaction of growing neurites with original synaptic sites in the process of neuromuscular regeneration.